162. FRIEND OR FOE? THE ROLES OF PAF AND p53 DURING EMBRYONIC DIAPAUSE

2010 ◽  
Vol 22 (9) ◽  
pp. 80
Author(s):  
J. C. Fenelon ◽  
C. O'Neill ◽  
G. Shaw ◽  
M. B. Renfree
Keyword(s):  
Tammar Wallaby ◽  
Early Embryo ◽  
Human Reproduction ◽  
Embryonic Diapause ◽  
Embryonic Cells ◽  
Active Growth ◽  
Molecular Control ◽  
Before And After ◽  
P53 Mrna

In the tammar wallaby, Macropus eugenii, the blastocyst normally remains in embryonic diapause for 11 months without cell division or apoptosis occurring. Progesterone regulates reactivation by inducing active secretion from the endometrium, but the molecular cross-talk between the endometrium and blastocyst is unknown. This process may involve the phospholipid paf. Paf is an embryotrophin that acts as a trophic/survival factor in the early embryo, partly by inactivating (via the PI3K/Akt pathway) the expression of p53, a cell cycle arrest factor (1,2). In vitro, paf production from the tammar endometrium increases after diapause (3). This study examined the expression of the paf receptor (pafr) and p53 in the tammar endometrium and embryo at entry into, during and reactivation from diapause. Both pafr and p53 mRNA were expressed in the endometrium at all stages. However there was no quantitative change in pafr expression. In the endometrium, pafr protein is present on the membrane of the glandular epithelium at all stages examined, but p53 was not expressed in the endometrial nuclei at any stage and hence does not appear to be active. Both pafr and p53 mRNA were also expressed in the embryo from the early cleavage stages, during diapause and in the reactivated blastocyst. Pafr protein was present in the embryo both before and after diapause, but levels were greatly reduced during diapause, indicating it may be necessary for active growth. Unexpectedly, the expression of p53 in the embryo does not appear to depend on the presence or absence of pafr. p53 was expressed in the nuclei of the cleavage stage embryonic cells before diapause, but not during or after diapause. These results suggest that paf and pafr may participate in the molecular control of embryonic diapause in the tammar independent of p53. (1) Jin XL et al. (2009) Biology of Reproduction 80: 286–294.(2) O’Neill C (2005) Human Reproduction Update 11(3): 215–228.(3) Kojima T et al. (1993) Reproduction Fertility Development 5: 15–25.

239. Expression of PAF-R and p53 in the endometrium during entry into and reactivation from diapause in the tammar wallaby

2008 ◽  
Vol 20 (9) ◽  
pp. 39
Author(s):  
J. C. Fenelon ◽  
G. Shaw ◽  
M. B. Renfree
Keyword(s):  
Tammar Wallaby ◽  
Early Embryo ◽  
Human Reproduction ◽  
Embryonic Diapause ◽  
P53 Expression ◽  
Cellular Location ◽  
Macropus Eugenii ◽  
A Cell

Embryonic diapause is widespread amongst mammals, but is especially common in the kangaroos and wallabies. In the tammar, Macropus eugenii, the sequence of endocrine events leading to embryonic diapause and reactivation are well defined and the blastocyst can remain in diapause for up to 11 months without cell division or apoptosis occurring (Renfree and Shaw 2000). The ovarian hormones exert their effects on the blastocyst by alterations in the endometrial secretions, but the molecular cross-talk between the endometrium and blastocyst is unknown. One possible regulator of diapause is the phospholipid PAF, an embryotrophin that acts as a trophic/survival factor for the early embryo (O'Neill 2005) partly by inactivating the expression of p53, a cell cycle inhibitor, via the PI3-K pathway. PAF is released from the tammar endometrium around the time of reactivation from diapause (Kojima et al. 1993). This study examined the expression of PAF-R and p53 in the tammar endometrium at entry into, and reactivation from, diapause. PAF-R and p53 were highly conserved with orthologueues in human and mouse. PAF-R and p53 expression was assessed by RT–PCR and both genes were expressed in the endometrium at all stages examined. Quantitative PCR (QPCR) studies performed for PAF-R in the endometrium show that levels of PAF-R vary depending on the stage examined and appear to be increasing at entry into diapause and decreasing at exit from diapause. Immunohistochemical (IHC) studies are in progress to determine the cellular location of PAF-R in the endometrium and confirm the QPCR results. QPCR and IHC studies are in progress to determine if there is any change in levels of expression or cellular location of p53 between the stages examined and how this relates to PAF-R availability. These results suggest that the control of diapause in the tammar involves interactions between multiple factors. (1) Renfree MB, Shaw G (2000) Diapause. Annu Rev Physiol 62, 353–375 (2) O'Neill C (2005) The role of Paf in embryo physiology. Human Reproduction Update 11, 215–228 (3) Kojima T et. al. (1993) Production and secretion of progesterone in vitro and presence of PAF in early pregnancy of the marsupial, Macropus eugenii. Reproduction Fertility Development 5, 15–25.

Ovarian steroid metabolism and oestrogens in the corpus luteum of the tammar wallaby

1984 ◽  
Vol 101 (2) ◽  
pp. 231-NP ◽  
Author(s):  
M. B. Renfree ◽  
A. P. F. Flint ◽  
S. W. Green ◽  
R. B. Heap
Keyword(s):  
Corpus Luteum ◽  
Follicular Development ◽  
Hydroxysteroid Dehydrogenase ◽  
Tammar Wallaby ◽  
Steroid Metabolism ◽  
Interstitial Tissue ◽  
Corpora Lutea ◽  
Embryonic Diapause ◽  
Probable Source

ABSTRACT Ovaries were obtained from tammar wallabies at various stages of the reproductive cycle to examine the occurrence of oestrogens in corpora lutea, and the synthesis and metabolism of steroids in the corpus luteum and ovarian cortical and interstitial tissues. Corpora lutea contained oestradiol-17β and oestrone during embryonic diapause and at all stages of pregnancy studied after blastocyst activation. Aryl sulphatase, 3β-hydroxysteroid dehydrogenase and 17β-oxidoreductase were shown to be present in luteal and other ovarian tissues by incubation in vitro with labelled substrates. Aromatase was undetectable in corpora lutea or in interstitial tissue, but was present in the ovarian tissues (including follicles) which remained after removal of corpora lutea. The probable source of the oestrogens detected in the corpus luteum is discussed in relation to their role in the inhibition of follicular development during embryonic diapause. J. Endocr. (1984) 101, 231–240

scholarly journals Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.

1979 ◽  
Vol 83 (1) ◽  
pp. 143-158 ◽  
Author(s):  
E Frank ◽  
G D Fischbach
Keyword(s):  
Synapse Formation ◽  
Synaptic Cleft ◽  
Postsynaptic Membrane ◽  
Active Growth ◽  
Before And After ◽  
Nerve Muscle ◽  
Scanning Em ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.

scholarly journals Paf receptor expression in the marsupial embryo and endometrium during embryonic diapause

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 21-31 ◽  
Author(s):  
Jane C Fenelon ◽  
Geoff Shaw ◽  
Chris O'Neill ◽  
Stephen Frankenberg ◽  
Marilyn B Renfree
Keyword(s):  
Platelet Activating Factor ◽  
Tammar Wallaby ◽  
Receptor Expression ◽  
Limiting Factor ◽  
Embryonic Diapause ◽  
Paf Receptor ◽  
Sequential Activation ◽  
Uterine Environment ◽  
Low Levels

The control of reactivation from embryonic diapause in the tammar wallaby (Macropus eugenii) involves sequential activation of the corpus luteum, secretion of progesterone that stimulates endometrial secretion and subsequent changes in the uterine environment that activate the embryo. However, the precise signals between the endometrium and the blastocyst are currently unknown. In eutherians, both the phospholipid Paf and its receptor, platelet-activating factor receptor (PTAFR), are present in the embryo and the endometrium. In the tammar, endometrial Paf releasein vitroincreases around the time of the early progesterone pulse that occurs around the time of reactivation, but whether Paf can reactivate the blastocyst is unknown. We cloned and characterised the expression of PTAFR in the tammar embryo and endometrium at entry into embryonic diapause, during its maintenance and after reactivation. Tammar PTAFR sequence and protein were highly conserved with mammalian orthologues. In the endometrium, PTAFR was expressed at a constant level in the glandular epithelium across all stages and in the luminal epithelium during both diapause and reactivation. Thus, the presence of the receptor appears not to be a limiting factor for Paf actions in the endometrium. However, the low levels of PTAFR in the embryo during diapause, together with its up-regulation and subsequent internalisation at reactivation, supports earlier results suggesting that endometrial Paf could be involved in reactivation of the tammar blastocyst from embryonic diapause.

111 RISK OF CHLAMYDIA ABORTUS TRANSMISSION VIA EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 186
Author(s):  
F. Fieni ◽  
M. Oseikria ◽  
K. Laroucau ◽  
F. Vorimore ◽  
D. Tainturier ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Early Embryo ◽  
Control Group ◽  
Bovine Embryo ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Chlamydia Abortus ◽  
Zoonotic Risk

Chlamydia abortus (C. abortus) in cattle has been reported sporadically throughout the world and is implicated in respiratory, ocular, and reproductive disease as abortion, infertility, chronic mastitis, vaginal discharge, and endometritis. In addition, C. abortus presents a zoonotic risk exposure of pregnant women to infected animal and can lead to severe septicaemia in the mother, resulting in spontaneous abortion or stillbirth of the fetus. To investigate the risk of C. abortus transmission via bovine embryo transfer, our study aims to determine whether the embryonic ZP of in vitro-produced embryos protects early embryo cells against C. abortus infection and whether the bacteria adhere to or infect the cells of early bovine embryos (ZP-free) after in vitro infection. We also evaluated the efficacy of the washing procedure recommended by the IETS to decontaminate bovine embryos exposed to C. abortus in vitro. Ninety (8 to 16 cells) bovine embryos, produced in vitro, were randomly divided into 10 batches. Eight batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4.8 × 107 Chlamydia/mL of AB7 strain (ANSES, Maisons-Alfort, France). After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a PBS and 5% FCS solution without trypsin nor antibiotics in accordance with IETS guidelines. In parallel, 2 batches of 5 embryos (1 ZP-intact and 1 ZP-free) were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13 000 × g. The embryos and wash pellets were tested using RT-PCR. Chlamydia abortus DNA was found in all ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the tenth wash fluid for 1 batch (1/4) of ZP-intact infected embryos and in 3 batches (3/4) of ZP-free infected embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. These results demonstrate that C. abortus adheres to or penetrates the ZP as well as the early embryonic cells of in vitro-produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring. Nevertheless, the finding of C. abortus DNA by RT-PCR did not imply that the bacteria found is still infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. abortus would eliminate the bacteria from the ZP.

scholarly journals The Proteome of Equine Oviductal Fluid Varies Before and After Ovulation: A Comparative Study

2021 ◽  
Vol 8 ◽  
Author(s):  
Pablo Fernández-Hernández ◽  
Federica Marinaro ◽  
María Jesús Sánchez-Calabuig ◽  
Luis Jesús García-Marín ◽  
María Julia Bragado ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Absolute Quantification ◽  
Early Embryo ◽  
Fold Change ◽  
Ovulatory Follicle ◽  
Before And After ◽  
Nano Liquid Chromatography ◽  
Isobaric Tags ◽  
Oviductal Fluid

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro. Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle (n = 4) or a recent ovulation (n = 4); the samples were kept at −80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus. Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm–oviduct interaction, fertilization, and metabolism, among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses.

166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?

2013 ◽  
Vol 25 (1) ◽  
pp. 231
Author(s):  
A. Alsaleh ◽  
J. L. Pellerin ◽  
C. Roux ◽  
M. Larrat ◽  
G. Chatagnon ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Early Embryo ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Tissue Samples ◽  
Worldwide Distribution

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Detection of significant bacterial loads in flushing media and tissue samples (oviducts and uterine horns) from the genital tracts of nonpregnant goats is a risk factor for in utero infection and transmission during embryo transfer (Alsaleh et al. 2011 CIMID 34, 355–360). The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the washing protocol recommend by the IETS for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and PCR, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9–9, 11–11, and 4–4 in replicates 1, 2, and 3, respectively) were placed in 1 mL of MEM containing 107 C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37°C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3–3, 5–5, and 2–2 in replicates 1, 2, and 3, respectively) were submitted to the same procedures but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 h at 13 000g. The washed embryos and pellets were tested by PCR. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first 5 washing fluids for ZP-free embryos and in the first 8 washing fluids for ZP-intact embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly demonstrate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to cling to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

scholarly journals Misjudging early embryo mortality in natural human reproduction

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 702
Author(s):  
Gavin E. Jarvis
Keyword(s):  
Human Embryo ◽  
Expert Witness ◽  
Laboratory Data ◽  
Large Body ◽  
Early Embryo ◽  
Human Reproduction ◽  
Human Embryos ◽  
Natural Conditions ◽  
Embryo Mortality

In 2002, in a judgment relating to the use of the morning-after pill, Mr Justice Munby held that pregnancy begins with the implantation of an embryo into the uterus of a woman. The case involved a large body of expert witness evidence including medical and physiological details of human reproduction. Munby J. emphasised one particular aspect of this evidence: namely, the developmental failure rate of human embryos after fertilisation. Under natural conditions, embryo loss is approximately 10-40% before implantation, and total loss from fertilisation to birth is 40-60% (Jarvis, 2016). By contrast, and based on expert witness testimony, Munby J. stated that not much more than 25% of successfully fertilised eggs reach the implantation stage, and that fewer than 15% of fertilised eggs result in a birth, figures that do not accurately represent scientific knowledge regarding human embryo mortality and pregnancy loss under natural conditions. Rather, these figures were derived from experimental laboratory data and clinical outcomes from in vitro fertilisation treatment. Testimony provided by other expert witnesses directly contradicted these specific numerical claims. In emphasising these figures, Munby J. gave the impression that human embryo mortality is substantially higher than available scientific evidence indicated. In this critique, all the scientific expert witness evidence is presented and reviewed, and an explanation provided for why the emphasised figures are wrong. Whether there are implications of Munby J.’s scientific misjudgment on the legal outcome is for others to consider.

scholarly journals The blood vessels development, morphogenesis and blood circulation are three ontologic groups highly up-regulated in porcine oocytes before in vitro maturation

2017 ◽  
Vol 5 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Mariusz J. Nawrocki ◽  
Piotr Celichowski ◽  
Joanna Budna ◽  
Artur Bryja ◽  
Wiesława Kranc ◽  
...  
Keyword(s):  
Blood Vessel ◽  
Blood Vessels ◽  
Blood Circulation ◽  
In Vitro Maturation ◽  
Embryo Implantation ◽  
Early Embryo ◽  
Expression Of Genes ◽  
Before And After

AbstractThe mammalian oocytes undergo significant biochemical and structural modifications during maturation both in vitro and in vivo. These changes involve chromatin reorganization and modification within metabolic status of cytoplasmic organelles. After oocytes’ successful maturation the substantially increased storage of RNA was observed. Moreover, the early embryo interaction with maternal endometrial tissue after fertilization is up to now considered as the main marker of proper embryo implantation and early growth. In this study, we first investigated the expression profile of genes involved in blood vessel formation and blood circulation in porcine oocytes before and after in vitro maturation.The cumulus-oocyte complexes were collected from pubertal Landrace gilts and classified as before in vitro maturation (in Vivo) or after in vitro maturation (in Vitro). The RNA was isolated from these two experimental groups and analyzed using Affymetrix microarrays.We found an increased expression of genes involved in ontological groups such as “blood circulation” (TPM1, ECE1, ACTA2, EPHX2, EDNRA, NPR2, MYOF, TACR3, VEGFA, GUCY1B3), “blood vessel development” (ANGPTL4, CYR61, SEMA5A, ID1, RHOB, RTN4, IHH, ANGPT2, EDNRA, TGFBR3, MYO1E, MMP14), and “blood vessels morphogenesis” (ANGPT2, as well as other common transcripts) in in Vivo group as compared to decreased expression of these genes in in Vitro group of oocytes.It has been suggested that investigated genes undergo significant expression before in vitro maturation, when enhanced storage of large amount of RNA takes place. Creating templates for synthesis of proteins is required for formation of fully mature gametes and early embryo growth. Therefore we hypothesized that the processes of vascularization and/or angiogenesis reach a high activity in immature oocytes and are distinct from achievement of maturational stage by oocytes in pigs.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

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